Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions

Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of this non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb infusion did not suppress infectious viral titers in the lung as potently as NTD neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Finally, Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection in SARS-CoV-2 infection. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.


145
To further investigate the importance of Fc effector function, we also sought to up-148 wildtype Fc (Figure 2A). For both antibodies, DLE substitutions increased total binding to 149 mouse FcRs compared to wildtype G1m17 versions (Figure 2B-I

165
We next compared the ability of G1m17, LALA-PG, and DLE versions of both antibodies 166 to mediate antibody-dependent cellular phagocytosis (ADCP) of recombinant NTD. For use as a 167 negative control antigen for non-neutralizing NTD antibodies, we designed a modified Spike 168 NTD that eliminated binding of the non-neutralizing NTD antibodies. We solved the structure of 169 DH1052 Fab in complex with the Spike trimer via negative stain electron microscopy (NSEM) 170 and identified the loops at amino acids 70-76, 182-187, and 211-214 as candidate contact sites 171 on the NTD (Figure 3A and B). We produced three NTDs that mutated each loop individually 172 (NTD_ADEm1a-c) and one NTD that contained mutated versions of all three putative contact . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint

225
We sought to understand why LALA-PG mutant antibodies did not result in higher gross 226 lung discoloration scores despite higher virus replication. An overabundance of proinflammatory 227 cytokines in the lungs has been suggested to be one mechanism by which COVID-19 lung 228 damage occurs (27). Thus, we compared the lung cytokine profile four days after SARS-CoV-2 229 MA10 challenge in mice that received LALA-PG or wildtype NTD antibody treatments. We 235 Whereas proinflammatory cytokine IL-6 level was not significantly different between wildtype 236 DH1052 and isotype control, the DH1052_LALA-PG group showed a significant decrease in IL-237 6 expression compared to isotype (p<0.001) ( Figure 6A). Additionally, the DH1052_LALA-PG 238 administration markedly increased antiviral cytokines such as TNF, IL-12, IL-1, and IFN 239 compared isotype control (p<0.001) ( Figure 6A). DH1050.1_G1m17-administration did not 240 increase these cytokines to the same extent ( Figure 6B). Cytokines and chemokines 241 associated with T cell responses (IL-2, RANTES, MIP1 and MIP2) were also increased in the 242 LALA-PG group compared to DH1052_G1m17 ( Figure 6A). Overall, the cytokine response had 243 a Th1 bias with IFN, TNFa, IL-1 and IL-12 being elevated. Altogether, the Fc functional 244 knockout DH1052_LALA-PG led to an increase in antiviral cytokines relative to isotype control 245 treatment in vivo, with the increase being far larger than what G1m17 induced ( Figure 6A). This 246 response suggests in the absence of Fc-mediated activity, robust antiviral cytokine activity was 247 upregulated, and less IL-6 was released. This cytokine profile was associated with minimal 248 macroscopic lung discoloration despite high infectious titers (Figure 5C and 5E).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint

262
We hypothesized that if Fc effector functions mediated by DH1052 protected mice from 263 SARS-CoV-2, then enhancing the ability of the antibody to mediate Fc effector functions would 264 improve protection from SARS-CoV-2 infection. Our in vitro experiments showed that the DLE 265 substitutions markedly increased ADCC and also modestly increased ADCP allowing us to test 266 this hypothesis (Figures 2J and 5). We passively immunized mice and challenged them 267 following the same protocol as with G1m17 antibodies (Figure 5A). DH1052_DLE showed 268 significantly reduced viral titers compared to titers from DH1052_G1m17 mice (p = 0.016, 269 Wilcoxon Test, n=10) ( Figure 5C). The mice that received DH1052_DLE also had significantly 270 higher final body weights compared to the G1m17 group (p = 0.01) (Figure 5D), and all mice in 271 this group achieved lung discoloration scores of 0 ( Figure 5E). Thus, enhancing the Fc function 272 of a non-neutralizing NTD antibody improved protection from infection. Although DH1052 is a 273 non-nAb and DH1050.1 is a nAb, there were no significant differences in infectious virus titers in . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint 274 the lungs (p=0.10), body weights (p=0.71), or lung discoloration scores (p=1.00) between 275 groups of mice that received DLE versions of either of these two antibodies (Figures 5C-H).

283
With SARS-CoV-2 variants escaping from nAbs, the protection from severe disease 284 afforded by non-nAbs is a key question. Even though current COVID vaccines no longer protect 285 against transmission, they continue to protect against severe disease and death (28, 29, 30).
286 Our study shows that wildtype non-nAbs can protect against manifestations of clinical disease in 287 a mouse model. The mechanism of protection is Fc-mediated effector functions given that 288 antibodies with ablated Fc effector functions conferred no benefit over negative control antibody.
289 It should be noted that there were differences in the degree of protection by non-nAbs 290 compared to nAbs. More specifically, mice that received non-nAb showed initial weight loss that 291 subsided by day 2. This phenotype can be explained by the time it takes for initial infection of  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint 300 harness Fc effector functions to mediate protection (21, 22, 23). The difference in Fc effector 301 function requirement for antibody-mediated protection has been attributed to differences in 302 accessibility of the Fc region due to angles of approach (22, 31), but antibody neutralization 303 potency, binding stoichiometry to Spike, and antibody epitope specificity may also explain the 304 discordant results from different studies (6).

305
The antibody versions that lacked Fc engagement resulted in a large increase in 306 cytokine secretion. As the lung faces invading pathogens continuously, the sources of lung 307 cytokines has been intensely studied (32). This upregulation may reflect an increase in epithelial 308 cell TNF and IL-1 release in the presence of increased viral titers due to an absence of Fc-      373 Fab (orange) in complex with SARS-CoV-2 Spike 2P (gray). In the enlargement, the density . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint 526 electron microscope operated at 120 KV at 82,000x nominal magnification and captured with a 527 2k x 2k CCD camera at a pixel size of 4.02 Å. Three-dimensional reconstructions were 528 calculated with standard procedures using Relion 3.0 (46). Images were created using UCSF 529 Chimera (47).

551
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint 552 Luminex Cytokine assay 553 Lung homogenate protein concentrations were determined using a Bio-Rad DC protein assay 554 performed according to the manufacturer's protocol and read on a Synergy H1 plate reader 555 (Agilent). Concentrations were calculated by extrapolation from a BSA standard curve using 556 Gen5 v.3.00 (Agilent). Cytokines in undiluted homogenate were quantified using a 26-plex 557 Luminex bead array assay (ThermoFisher EPX260-26088-901) performed according to the  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 26, 2023. ; https://doi.org/10.1101/2023.07.25.550460 doi: bioRxiv preprint